Changes in intracellular pH of Physarum plasmodium during the cell cycle and in response to starvation

The intracellular pH of Physarum plasmodia was monitored under conditions of growth and during starvation by means of recessed tip pH microelectrodes. There is a cycle of intracellular pH that corresponds to the period of the cell cycle, with a low point at mid-interphase of pH 7.0 and a peak of pH 7.5 just at mitosis. Experiments in which the intracellular pH is artificially lowered suggest that there is a critical period 1 h before mitosis in which the pH must be high (>7.2), but that mitosis itself can proceed at lower values. During the process of differentiation induced by starvation the intracellular pH drops to very low values (6.6 by 15 h) and refeeding can quickly reverse this condition and restore the pH cycle and nuclear division. Additionally, artificially lowering the intracellular pH will give rise to morphology which resembles the first stages of starvation-induced differentiation.     

A purified cellular extract accelerates the cell cycle in Physarum polycephalum

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.     

Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

The ultrastructure of mitosis in myxamoebae and plasmodia of Physarum flavicomum

Electron microscopy of glutaraldehyde-osmium-fixed samples of haploid myxamoebae and diploid plasmodia of the myxomycete Physarum flavicomum Berk. reveal dissimilar spindle apparatus during mitosis in the two cell types. Myxamoebae exhibit an astral type of mitosis with centrioles at the poles and nuclear envelope breakdown during prophase. Plasmodial nuclei lack centrioles at mitosis and have an intranuclear spindle, with nuclear envelope persisting during the entire division. Coated vesicles are noted during prophase and telophase in myxamoebae and their role in spindle formation and dispersion is suggested.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

No growth cycle in Physarum?

The activities of a number of enzymes have been determined in growing plasmodia of Physarum polycephalum at 1 h intervals during the naturally synchronous nuclear division cycle. The enzymes selected represent the main pathways of energy metabolism, they do not require posttranslational steps for activation, nor are they directly involved in DNA replication, mitosis or differentiation. We found constant activity levels for all enzymes tested and discuss the lack of endogenous oscillations in the synthesis of the corresponding proteins with respect to the growthcycle concept.     

Demonstration of different patterns of microtubule organization in Physarum polycephalum myxamoebae and plasmodia using immunofluorescence microscopy

We have used anti-tubulin antibodies and immunofluorescence microscopy to determine the overall distribution of microtubules during interphase and mitosis in both the myxamoebae and plasmodia of the slime mold Physarum polycephalum. We have paralleled these observations with electron microscopy of the same stages. The myxamoebae possess a network of cytoplasmic microtubules whilst the coenocytic plasmodium does not possess any cytoplasmic microtubules--at either interphase or mitosis. In plasmodia microtubules are, however, elaborated by an intranuclear microtubule organizing centre (MTOC) during prophase of mitosis and these microtubules proceed to form part of the mitotic spindle. There is little difference in the overall distribution and arrangement of microtubules during division of either the myxamoebal or plasmodial nuclei. These findings are discussed in relation to the synthesis of tubulin during the plasmodial cell cycle and the rearrangements of the nuclear envelope during mitosis.     

A Comparative Study of Mitosis in Amoebae and Plasmodia of the True Slime Mold Didymium nigripes

SYNOPSIS. Studies comparing mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes reveal that at the time of differentiation pronounced changes occur in the mitotic process. Not only does the amount of time required for division of the 2 stages differ, but plasmodial mitosis is characterized by persistence of the nuclear membrane and the apparent lack of centrioles. The origin of multinucleate plasmodia from uninucleate cells which have already undergone cytoplasmic differentiation is described. Division time in a population of amoebae becomes more uniform after those cells which are destined to form plasmodia have differentiated.
The observations and data presented indicated that differences in mitotic behavior also occur between amoebae of 3 stocks with differences in plasmodial structure and behavior. Comparison of mitosis in the plasmodia of these 3 stocks revealed no significant differences.     

Isolation of cell cycle mutants of Physarum polycephalum

Summary Asynchronous amoebal cultures of temperature-sensitive mutants of Physarum polycephalum were examined cytologically, and two cell cycle mutants were identified. Genetic analysis indicated that each mutant carried a single mutation that was expressed in both amoebal and plasmodial phases. Thus it is possible to isolate cell cycle mutations expressed in plasmodia by initial isolation and analysis of amoebal mutants, a quicker procedure than the alternative of isolating plasmodial mutants directly. The two mutants were studied further by measuring nuclear DNA contents and synthesis of macromolecules. Both mutants gave results consistent with a block in nuclear division.     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.     

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

Nuclear membrane behavior during mitosis in normal and heteroploid Myxomycetes

Summary Photomicrographic evidence is presented of the difference in behavior of nuclear membranes during mitosis in amoebae, zygotes and plasmodia of Myxomycetes. One of the species was cultured on bacteria and possessed a normal cycle of plasmogamy and karyogamy between the amoebal and plasmodial phases. The second species was axenically grown in liquid media and had become highly heteroploid and lacked the ability to develop into plasmodia, existing only in the amoeboid form. The significance of the amoeboid form of mitosis in the heteroploid axenically cultured strain is discussed in relation to the difference in nuclear membrane behavior and the possible significance of such behavior.     

The effects of anaerobiosis and uncouplers of oxidative phosphorylation on mitotic timing and the induction of intranuclear macrotubules in the slime mold,Physarum polycephalum

Summary Mitotically synchronous plasmodia of the slime moldPhysarum polycephalum were subjected to brief exposures of either pure atmospheres of carbon dioxide or nitrogen gases or to pulsetreatments with respiratory poisons (sodium azide, sodium arsenate, or 2,4-dinitrophenol, DNP) at many different phases of the mitotic cycle to assess their effects on the mechanism(s) controlling the timing of mitosis. Plasmodia were fully viable after a pulse of CO₂ lasting up to 90 minutes or after a N₂-pulse of 30 minutes in duration. Upon return to normal aeration, all treated plasmodia entered a fully synchronous mitosis with a variable excess mitotic delay, which was dependent on the duration of the pulse and time of application in the mitotic cycle. Likewise, plasmodia exposed to 15-minute-pulses of a sublethal dose of sodium arsenate (0.1 mM), sodium azide 0.05 mM) and 2,4-DNP (0.2 mM) yield characteristic patterns of excess mitotic delay upon returnal to normal culture conditions. Two different types of phase response curves (PRC) were generated by these treatments. This suggests that at least two distinct respiratory-linked physiological mechanisms are involved in control of mitosis onset and regulation of mitotic timing inPhysarum.Electron microscope observations of CO₂-treated plasmodia reveal the induction of intranuclear 40–60 nm diameter macrotubules at all stages of the G₂ phase up to and including prometaphase. Both anoxia and sodium azide treatments are effective in macrotubule induction, and both reversibly disrupt the normal tubular cristae organization of mitochondria. In early G₂, macrotubules polymerize in association with both the inner membrane of the nuclear envelope and the nucleolus, while the tubule-organizer region, TOR, serves as the only nucleating site for macrotubules in late G₂ nuclei, coincident with the onset of mitosis and TOR formation.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Cyclic fluctuations of the mitotic Ca2+-ATPase during the cell cycle of the myxomycete Physarum polycephalum

The occurrence of the mitotic Ca2+-ATPase, resembling the enzyme described for higher organisms, is demonstrated in multinuclear plasmodia of the myxomycete Physarum polycephalum. The activity of this enzyme undergoes cyclic fluctuations during the synchronous nuclear cycle with a minimum in early G2-phase and a maximum around the time of mitosis.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

The nuclear division and parasexual cycle inPenicillium

The vegetative nuclear division inPenicillium differs from classical mitosis, and a model for the division process is presented. In early divisional stages the interconnected chromosomes are arranged in a ring which breaks, giving rise to a linear configuration which divides by longitudinal splitting. The break may occur with equal probability between each of the chromosomes. At the end of the division process the daughter nuclei regain ring structure. One of the chromosomes is believed to represent a separation center for all the chromosomes. In diploid strains the two haploid genomes show a close somatic association and the nuclear configurations occurring during the divisional cycle are identical to those in the haploids. The double break of the ring structures in diploids will give recombinant nuclei in certain cases. The model explains the available data of the parasexual cycle, and both diploidization and haploidization are believed to represent singlestep processes.     

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.